Project Summary - High throughput multiplexed assay for profiling gene expressions of schizophrenia iPSC-derived neurons This proposal is to develop and validate a secondary high-throughput screening (HTS) assay for new therapeutics for schizophrenia (SCZ). By assessing primary hit compounds? ability to ameliorate transcriptional dysregulation in SCZ neurons and not to dysregulate normal neurons in 384-well format, the potential mechanism of actions (MOAs) of the hits can be quickly assessed in a high-throughput and low cost fashion. This assay addresses the main shortcoming of phenotypic screens: hits of unknown mechanisms. We have a validated primary HTS synaptic density assay using forebrain glutamatergic neurons (FGNs) derived from induced pluripotent stem cells (iPSCs) bearing a Disrupted in schizophrenia 1 (DISC1) frameshift mutation. This proposed secondary screen, our primary screen and other confirmatory assays we have developed form a powerful toolset for SCZ drug discovery. SCZ, a major public health problem, affects around 1% of the world?s population. It manifests with diverse symptoms, is challenging to treat, has complex genetic risk associations and poorly understood etiology. 30% of patients do not respond to medications, 30% show partial response, and only 30% enter full remission. SCZ medications tend to be partially effective for positive symptoms but have limited or no effect for negative symptoms or cognitive impairments, which are the most important predictors for long-term social functioning. Hence, new psychotropic therapeutics are need to address significant unmet medical needs. We have optimized differentiation protocols that produce highly pure populations of cortical neural progenitors (NPCs), GABAergic interneurons and FGNs (>99%, >80% and >90% respectively). The purity of differentiated populations allows us to confidently attribute assay readouts to intrinsic properties of the cellular models and experimental manipulations. We have done high-fidelity RNAseq of the transcriptome of SCZ FGNs, enabled by high-purity iPSC differentiation. To assess changes in expressions of the dysregulated genes when treated with hit compounds, we adapt the RNA-mediated oligonucleotide Annealing Selection and Ligation with next-gen sequencing (RASLseq) technology. Our team has optimized assay conditions and we can quantitatively profile the expression level of 200-300 genes in each well of the 384-well plate. With our work-to-date, we don?t anticipate technical difficulties achieving the following aims: Aim 1. Design and test probes for 400 dysregulated genes. Based on RNAseq of SCZ FGNs, pathway analyses and functionality assessment, we chose 200 upregulated and 200 downregulated genes to profile. Aim 2. Validate assay with FGNs derived from 15 SCZ-patient specific iPSC lines. We will quantify the expression of the 400 genes plus house-keeping gene controls in FGNs derived from 15 SCZ iPSC lines, and compare to available RNAseq data. Aim 3. Validate assay with hits from synaptic density primary screen with NINDS Custom Collection 2, which consists of 1040 compounds (known to influence brain activity, 75% FDA approved). Assess if hits ameliorate expression dysregulation in DISC1 FGNs and if hits do not dysregulate expressions in isogenic normal control FGNs. We will further assess if the impact on gene expression is consistent with their respective MOAs, therapeutic and side effects.